PROJECT SUMMARY Cancer cells have been shown to be sensitive to apoptotic stimulus of tumor necrosis factor ?-related apoptosis-inducing ligand (TRAIL), whereas normal cells showed very little response. TRAIL was shown to be active as a single agent and exhibited synergistic activity with certain chemotherapeutic agents or radiotherapy, causing marked regression or complete remission of tumors. There is increasing evidence that membrane-bound TRAIL expressed on the surface of activated T-lymphocytes can enhance T-cell effector function and augment T-cell tumoricidal activity. The ability to genetically engineer primary T-cells creates new and highly promising prospects for tumor immunity and cancer treatment. The transduction of T-cells with genes encoding chimeric antigen receptors enables T-cell recognition of antigens that are either poorly immunogenic or ignored by the immune system. In addition, the genetically engineered expression of therapeutic ligands (e.g. TRAIL) by T-cells can potently increase their tumoricidal activity. New strategies for tumor sensitization to TRAIL-based immunotherapies and modulation of TRAIL resistance are being developed and some can be translated to the clinic. Our central theme and hypothesis is that TRAIL overexpression by T- cells results in augmented apoptosis in tumor cells and that radiation and/or chemotherapy positively affect TRAIL-mediated tumor apoptosis during T-cell adoptive immunotherapy and can be used as a synergistic approach to enhance T-cell tumor targeting and effector function. In this application we propose to develop and test a novel theranostic approach to augment tumor apoptosis in a prostate cancer model with heterogeneous PSMA levels using TRAIL overexpression by chimeric antigen receptor (CAR)-grafted PSMA-specific T-cells. We will assess the effect of membrane-bound vs. secretable forms of TRAIL expressed by PSMA-specific T-cells on their ability to kill PSMA-positive and -negative targets. We will determine whether radiation/chemotherapy-induced ?sensitization? of TRAIL-resistant tumor cells correlates with an improved T-cell tumoricidal function. Ultimately, we will assess the feasibility and sensitivity of PET imaging to monitor PSMA-specific T-cell activation and delivery of TRAIL therapeutic payloads to the tumor. These processes will be monitored using multi-reporter gene and conventional imaging by assessing T- cell tumor targeting and activation as well as tumor response. Our proposal complements on-going clinical studies at MSK and other cancer centers by exploring new strategies designed to enhance T-cell effector function and improve treatment response. The results will provide preclinical support and justification to move toward clinical application in patients with prostate and other cancers undergoing immunotherapy with genetically modified T-cells. !